Cell suspension preparation technique and device

ABSTRACT

The present invention provides a unique method and or device suitable for producing a transplantable cellular suspension of living tissue suitable for grafting to a patient. In applying the method and or in using the device donor tissue is harvested, subjected to a cell dissociation treatment, cells suitable for grafting back to a patient are collected and dispersed in a solution that is suitable for immediate dispersion over the recipient graft site.

[0001] This application claims priority to Australian Provisional PatentApplication PR2989, filed Feb. 7, 2001, and U.S. Provisional PatentApplication Serial No. 60/281,527, filed Apr. 4, 2001, which areincorporated herein by this reference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates to a simple, rapid and cost effectivetechnique for grafting of cells, and in particular to a device forpreparing a suspension of cells from a tissue sample obtained from adonor site and applying that suspension of cells to a recipient site.

BACKGROUND ART

[0003] There are many methods of treating wounds known to those skilledin the art. For example, skin grafting techniques exist which aim toreconstruct the skin covering areas of the body where there is eitherdamage or defects to the skin. In general, these types of grafts areclassified according to their host-donor relationship and by theirthickness. The most clinically applied graft is the autologous graft,whereby tissue is taken from one area of the body and applied to anotherarea. The grafted tissue then develops a new blood supply and attachesto the underlying tissues.

[0004] There are several types of skin grafts presently used, includingsplit-thickness, full-thickness grafts, and micro-grafting. Each ofthese graft types must be prepared using certain techniques, and eachone has its inherent advantages and disadvantages. Split-thicknessgrafts often require considerable skill, time and expensive equipment toperform. Further, donor sites are painful, result in scarring and limitthe area able to be covered. Although they may be more successful thanfull-thickness grafts, they are usually less cosmetically attractive.Full-thickness grafts require less skill or expensive equipment, andtheir cosmetic appearance is better than that of split-thickness grafts.However, full-thickness grafts do not “take” as well as split-thicknessgrafts. Micro-grafts are more easily accomplished and require no specialinstruments. However, their cosmetic appearance is not as good as othertechniques, as the resulting scarring is unacceptable.

[0005] A variation to the above grafting techniques is the mesh graft,which is a type of split-thickness or full-thickness skin graft in whichparallel rows of slits are cut in the tissue being treated. Some of theadvantages of mesh grafts include: a greater coverage of the effectedarea, drainage of blood or serum from under the graft, and increasedconformity of the graft to uneven recipient areas. This technique hasbeen very successful, with 90 to 100 percent “take” when the grafts havebeen applied on healthy granulation beds.

[0006] An alternative to split-skin grafting is to form a blister undersuction at a donor site and transplant to the recipient site. Theproduction of blisters to treat wounds has been used since the 1960s.The blisters are produced by a suction device, such as Dermavac™, at asuction pressure of approximately 250-300 mmHg for 1-2 hours. Theblisters are then cut off and placed on the wound. The healing time isaround 10-14 days. There are several disadvantages of this method suchas the amount of time required to prepare the graft is too long and thegraft may not result in re-pigmentation of the area; or unevenpigmentation is common around the edges of the area of treatment.

[0007] Micro-grafting has become a more common approach for large areacover and involves the “snipping off” of a number of very small sectionsof tissue from a donor site and applying then to a dressing, which is inturn applied to the wound area.

[0008] The most advanced technology for the generation of a tissue invitro is to culture epidermis. Cultured epithelial autografts (CEA) arean important adjunct in the coverage of burns and other situations inwhich large areas of the body's surface experience skin loss. There aremany centres throughout the world with tissue culture facilities whoseaim is to produce autologous epithelial grafts for use in a wide varietyof applications. The usefulness and application of CEA is related to itsability to achieve confluent cells sheets suitable for grafting. Thistechnique overcomes many of the disadvantages of the previous treatmentsdescribed above. For example, cultured epithelial autografts reduce thedemand for donor sites. However, these autografts are slow growing andrequire time for culturing of the grafts, which often exceeds the timeof preparation of the recipient's sites.

[0009] The present invention provides a cellular suspension togetherwith a method for preparing that suspension and a device for itspreparation each of which seek to ameliorate one or more of thedisadvantages associated with prior art grafting technology.

SUMMARY OF THE INVENTION

[0010] The subject invention relates to a unique cell suspension andmethod for its preparation that is rapid, efficient and simple toprepare and apply. It also relates to a method for treating a patientusing the unique cell suspension and to an apparatus suitable for use inthe method preparation. Use of the described device while not essentialfor practising the method of the invention has been found tosignificantly reduce the complexity associated with the use ofconventional grafting technology such as CEA.

[0011] According to a first aspect of the invention there is provided amethod for preparing a cell suspension suitable for application to apatient, which method comprises the steps of:

[0012] (a) subjecting a tissue sample including cells suitable forgrafting to a patient, to at least a physical and or chemicaldissociating means capable of dissociating cellular stratum in thetissue sample;

[0013] (b) removing the tissue sample from the presence of the tissuedissociating means used in step (a) and harvesting in the presence of anutrient solution cells from the tissue sample, cells suitable forgrafting on to a patient wherein the nutrient solution is (i) free ofxenogenic serum, (ii) capable of maintaining the viability of the cellsuntil applied to a patient and (iii) is suitable for direct applicationto a region on a patient undergoing tissue grafting; and

[0014] (c) filtering the cellular suspension produced according to step(b) to remove large cellular conglomerates.

[0015] In a preferred form of the invention the dissociating means is ofa chemical nature such as an enzyme which is capable of disruptingcellular bonding like for example trypsin. Further, preferably thefiltered cellular suspension is diluted to an appropriate cell densityusing a nutrient solution, which may be anything from a basic saltsolution to a more complex nutrient solution.

[0016] According to a second aspect of the invention there is provided acell suspension produced according to the above method. Preferably thecells in the suspension are autologous cells (i.e. they are isolatedfrom the patient requiring an autograft). The inventors have observedthat by removing xenogenic serum from the cell suspension there is lesslikelihood of transmission of infection and xenogenic reactions betweena patient and the serum are eliminated. Another feature of the cellsuspension produced according to the above method is that the tissuesample used to isolate the cells in the suspension is removed from theenzyme solution before the cells are harvested. When cells are exposedto enzymes capable of damaging inter-cellular adhesion the viability ofthe cellular suspension decreases over time thereby reducing theefficiency of the grafting when applied to a patient. The cellsuspension produced according to the above method has been observed topossess greater cellular viability compared to comparative methods thatharvest the cells at regular intervals whilst the tissue is immersed inthe presence of enzymes like trypsin.

[0017] According to a third aspect of the invention there is provided amethod of treating a patient in need of graft surgery, said methodcomprising the steps of:

[0018] (a) preparing an cell suspension according to the above method;

[0019] (b) administering the suspension directly to a region on thepatient that requires a cell graft in a manner that facilitatesspreading of the cell suspension in a relatively even distribution overthe graft region.

[0020] In an alternate form, the invention consists in the use of acellular suspension suitable for grafts, which suspension is preparedaccording to the following steps:

[0021] (a) subjecting a tissue sample including cells suitable forgrafting to a patient, to an enzyme suitable for dissociating cohesivepieces of the tissue stratum in the sample;

[0022] (b) removing the sample from the enzyme solution used in step (a)and harvesting in the presence of a nutrient solution cells from thetissue sample, which cells are suitable for grafting on to a patientwherein the nutrient solution is (i) free of xenogenic serum, (ii)capable of maintaining the viability of the cells until applied to apatient and (iii) is suitable for direct application to a region on apatient undergoing tissue grafting;

[0023] (c) filtering the cellular suspension produced according to step(b) to remove large cellular conglomerates;

[0024] for the preparation of medicament suitable for the treatment oftissue disorders requiring grafting.

[0025] According to a fourth aspect of the invention there is providedan apparatus for developing a tissue regeneration solution, saidapparatus comprising:

[0026] (a) a heating means suitable for heating an enzyme solution to arequired temperature and which is capable of maintaining that solutionat the desired temperature for a suitable amount of time; and

[0027] (b) a filter recess comprising a filter means capable ofseparating large cellular congregates from a cellular suspension.

[0028] When the enzyme solution is placed in contact with the heatingmeans it is preferably heated in a manner that avoids localised heatingwithin the solution.

[0029] Other aspects and advantages of the invention will becomeapparent to those skilled in the art from a review of the ensuingdescription, which proceeds with reference to the following illustrativedrawings.

BRIEF DESCRIPTION OF DRAWINGS

[0030]FIG. 1 illustrates a perspective view of the apparatus with thelid open and the second member in place.

[0031]FIG. 2 illustrates a perspective view of the apparatus with thelid open and the second member removed and inverted.

[0032]FIG. 3a illustrates a perspective view of the first member of theapparatus.

[0033]FIG. 3b illustrates a perspective rear view of the first member ofthe apparatus.

[0034]FIG. 4 illustrates a perspective view of the base of theapparatus.

DETAILED DESCRIPTION OF THE INVENTION

[0035] Those skilled in the art will appreciate that the inventiondescribed herein is susceptible to variations and modifications otherthan those specifically described. It is to be understood that theinvention includes all such variation and modifications. The inventionalso includes all of the steps, features, compositions and compoundsreferred to or indicated in the specification, individually orcollectively and any and all combinations or any two or more of thesteps or features.

[0036] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended for thepurpose of exemplification only. Functionally equivalent products,compositions and where appropriate methods are clearly within the scopeof the invention as described herein.

[0037] Throughout this specification and the claims that follow, unlessthe context requires otherwise, the word “comprise”, or variations suchas “comprises” or “comprising”, will be understood to imply theinclusion of a stated integer or group of integers but not the exclusionof any other integer or group of integers.

[0038] Having regard to the above, this invention provides a uniquemethod and or device suitable for producing a transplantable cellularsuspension of living tissue suitable for grafting to a patient. Inapplying the method and or in using the device donor tissue isharvested, subjected to a tissue dissociating means, cells suitable forgrafting back to a patient are collected and dispersed in a solutionthat is suitable for immediate dispersion over the recipient graft site.

[0039] The subject invention has many advantages over the prior art someof which are illustrated in the following paragraphs.

[0040] 1. It provides a time efficient method for supplying a cellularcover to a tissue in a clinical setting. That is, cells are availablewhen needed at the time of surgery. This is achievable because there isa very short preparation period of the cells, thus allowing grafting tobe performed peri-operatively or in the rooms of a specialist physicianor General Practitioner.

[0041] 2. It provides a method and an apparatus which significantlyreduces the complexity associated with the use of conventional CEA's andis particularly useful in cases of burn injury that have presented late.In some instances, cells are unavailable at the time of surgery, eitherdue to delayed referral of a patient with an unhealed burn or simplybecause the time needed for culturing of the grafts had exceeded thatfor preparation of the recipient would bed. The present inventionameliorates the issue of graft preparation time.

[0042] 3. It aids in the achievement of rapid cell coverage in areas ofinjury and donor sites. It provides a means for reducing the size ofdonor sites—the biopsy donor site is markedly smaller than a split skingraft donor site and reduces or eliminates the use of split skin graftdonor sites; improves expansion rate of cell coverage; improves the rateof healing of small burns; is useful for small areas of skinreconstructions, such as scars; and improves scar quality.

[0043] 4. It ameliorates problems associated with the use of solutionsused during conventional tissue culture process. According to the methodof preparation and treatment the cells used in a graft are suspended ina nutrient solution free of xenogenic serum. That suspension is thenplaced directly onto the recipient site.

[0044] 5. It provides a means for the treatment of various skindisorders or diseases. For example, it may be used for the following:epidermal resurfacing, replacement after skin loss, site match-up duringre-pigmentation of an area of skin, treatment of burn wounds,leukoderma, vitiligo, piebaldism, in the treatment of scars—for example,caused through incorrect wound healing, improper scar direction or scardistortion from wound contraction, acne scars; resurfacing cosmeticdermabrasion, resurfacing after laser treatment and in association withdermal reconstruction. Additionally the method may be used for cellreplacement therapy, including, for example, nerve cell replacementtreatment, epithelial cell (such as urothelial cell, buccal mucosal celland respiratory epithelial cell) replacement treatment, endothelial cellreplacement treatment and osteogenic precursor cell replacementtreatment.

[0045] 6. It provides a means to produce a suspension of cells in aratio to each other comparable with those seen in situ. That is, due tothe manner of preparation of the cellular suspension, cells such askeratinocyte basal cells, Langerhans cells, fibroblasts and melanocytestypically have enhanced survival rates in comparison to standard tissueculture techniques, whereby selective cell culture can result in theloss of certain cell types. This has the advantage of allowing for thecorrect re-pigmentation of skin after a skin graft.

[0046] 7. It allows faster surgery and healing—thereby reducing traumafor patients during the phase of their medical care.

[0047] According to the first aspect of the invention there is provideda method for preparing a cell suspension suitable for use in resurfacingand regeneration of damaged tissue.

[0048] According to this method, tissue (preferably of an autologousnature) is harvested from a patient by means known in the art of tissuegrafting. Preferably this is achieved by taking a tissue biopsy. Withthe harvesting of the biopsy consideration must be given to the depth ofthe biopsy and the surface area size. The depth and size of the biopsyinfluence the ease at which the procedure can be undertaken and speedwith which a patient recovers from the procedure. In a highly preferredform of the invention the donor site should be chosen to appropriatelymatch the recipient site, for example post-auricular for head and neck,thigh for lower limbs, inner-upper-arm for upper limbs, or palm for soleor vice-versa.

[0049] Once a biopsy has been harvested from a patient the tissue sampleis subjected to physical and or chemical dissociating means capable ofdissociating cellular stratum in the tissue sample.

[0050] Methods for dissociating cellular layers within the tissues arewell known in the field. For example, the dissociating means may beeither a physical or a chemical disruption means. Physical dissociationmeans might include, for example, scraping the tissue sample with ascalpel, mincing the tissue, physically cutting the layers apart, orperfusing the tissue. Chemical dissociation means might include, forexample, digestion with enzymes such as trypsin, dispase, collagenase,trypsin-edta, thermolysin, pronase, hyaluronidase, elastase, papain andpancreatin. Non-enzymatic solutions for the dissociation of tissue canalso be used.

[0051] Preferably, dissociation of the tissue sample is achieved byplacing the sample in a pre-warmed enzyme solution containing an amountof enzyme sufficient to dissociate cellular stratum in the tissuesample. This may be achieved, for example using a trypsin solution,however, any other enzyme such as dispase, collagenase, trypsin-edta,thermolysin, pronase, hyaluronidase, pancreatin, elastase and papainthat cause cells to become detached from other cells or from solidsurfaces may be used for this purpose. When the enzyme used is trypsinthe enzyme solution used in the method is preferably calcium andmagnesium free. One such solution is preferably calcium and magnesiumion free phosphate buffered saline.

[0052] Where the tissue biopsy is derived from a patient's skin(comprising epithelialdermal cells) the amount of trypsin that might beused in the method is preferably between about 5 and 0.1% trypsin pervolume of solution. Desirable the trypsin concentration of the solutionis about 2.5 to 0.25%, with about 0.5% trypsin being most preferred.

[0053] The time period over which the tissue sample is subjected to thetrypsin solution may vary depending on the size of the biopsy sampletaken. Preferably the tissue sample is placed in the presence of thetrypsin solution for sufficient time to weaken the cohesive bondingbetween the tissue stratum. For example, where the tissue sample istaken from a patient's skin the sample might be placed in trypsin for a5 to 60 minute period. Preferably, the tissue sample is immersed in thetrypsin solution for between 10 and 30 minutes with 15 to 20 minutesbeing optimal for most tissue samples.

[0054] After the tissue sample has been immersed in the trypsin solutionfor an appropriate amount of time, the sample is removed from thetrypsin and washed with nutrient solution. Washing the tissue sample mayinvolve either partial or complete immersion of the treated sample inthe nutrient solution. Alternatively, and more preferably, the washsolution is dripped on the tissue sample in sufficient volume to removeand or significantly dilute any excess trypsin solution from the surfaceof the sample. Preferably any dilution that might occur would lead toless than 0.05% trypsin in the nutrient solution.

[0055] The nutrient solution used in the method should be capable ofsignificantly reducing and more preferably removing the effect of thetrypsin either by dilution or neutralisation. The nutrient solution usedin the method will also preferably have the characteristics of being (i)free of at least xenogenic serum, (ii) capable of maintaining theviability of the cells until applied to a patient, and (iii) suitablefor direct application to a region on a patient undergoing tissuegrafting. The solution may be anything from a basic salt solution to amore complex nutrient solution. Preferably, the nutrient solution isfree of all serum but contains various salts that resemble thesubstances found in body fluids; this type of solution is often calledphysiological saline. Phosphate or other non-toxic substances may alsobuffer the solution in order to maintain the pH at approximatelyphysiological levels. A suitable nutrient solution that is particularlypreferred is Hartmann's solution.

[0056] After application of the nutrient solution to the tissue sample,the cellular stratum of the sample are separated permitting cellscapable of reproduction to be removed from the cellular material andsuspended in the nutrient solution. Where the tissue sample is skin thedermis and epidermis are preferably separated to allow access to thedermal-epithelial junction of both surfaces.

[0057] Cells capable of reproduction are then removed from the separatedstratum by any means known in the art. Preferably, the reproductivecells are scraped off the surface of the stratum using an instrumentsuch as a scalpel. Cells capable of reproduction within thedermal-epithelial junction include but are not limited to keratinocytebasal cells, Langerhans cells, fibroblasts and melanocytes. Followingrelease of the cells from the tissue sample they are suspended in thenutrient solution. Preferably only a small volume of nutrient solutionis applied to the tissue sample during this harvesting step otherwisethe suspension may become too fluid therein providing difficulties inapplying the suspension to the graft.

[0058] To avoid excessively large cellular congregates in the cellularsuspension the suspension is preferably filtered. Any filter capable ofseparating excessively large cellular congregates from the suspensionmay be used in this preferred step of the invention. In a highlypreferred form of the invention the filter size is between 50 μm and 200μm. More preferably it is between 75 μm and 150 μm, with 100 μm beingone specific example.

[0059] Prior to application to the graft site or immediately afterfiltering, the cellular suspension may be diluted to produce anappropriate cell density suitable for the purpose to which thesuspension is to be used.

[0060] According to the second aspect of the invention there is providedan aqueous cell suspension produced by the method described in the firstaspect of the invention. The cell suspension provided by this method ishighly suitable for tissue regeneration and grafting techniques. Animportant advantage of utilising such a suspension in graftingtechnology is that it can be used to greatly expand the area or volumeof a wound that can be treated quickly by in situ multiplication of alimited number of cells. The number and concentration of cells seededonto graft site may be varied by modifying the concentration of cells insuspension, or by modifying the quantity of suspension that isdistributed onto a given area or volume of the graft site.

[0061] By suspending cells in a nutrient solution which is at least (i)free of xenogenic serum, (ii) capable of maintaining the viability ofthe cells until applied to a patient and (iii) is suitable for directapplication to a region on a patient undergoing tissue grafting, theinventors have found that the outcome of patient grafts is improved. Apartial explanation for this appears to be attributable to the removalof xenogenic serum and more preferably all serum from the cellsuspension. Xenogenic serum is a common additive in grafting culturemedium and is well known to cause potential infective andhypersensitivity problems. Such serum is however generally required forthe in vitro expansion of the cells and to neutralise the action of theenzyme if the enzyme used is trypsin. The nutrient solution used in thepresent invention does not require such serum because the cellpopulation within the suspension is not expanded prior to application tothe graft site. Rather cellular multiplication is encouraged on thepatient rather than in an in vitro system. When trypsin is usedneutralisation is achieved by other means.

[0062] Another unique feature of the cell suspension produced accordingto the method of the first aspect of the invention is that thecomposition of cells in the cellular preparation is comparable to thatseen in situ compared to prior art cellular preparation. One possibleexplanation for this is that in the prior art, culture of the cellularpreparation utilises selective culture for keratinocytes, therefore lossof cellular constituents such as fibroblasts and melanocytes occurswhereas the cellular suspension produced from the first aspect of theinvention has a cell composition comparable to the in situ cellpopulation. Another feature of the cellular suspension produced from thefirst aspect of the invention is that the graft cells are more viable asthey are harvested in a nutrient solution as distinct from prior artcell harvesting procedures which utilise techniques where the cells areharvested whilst exposed to powerful digestive enzymes for excessiveperiods of time. When the cells are exposed to such enzymes forexcessive periods of time the viability of the cellular suspensiondecreases.

[0063] According to the third aspect of the invention there is provideda method of treatment of the patient requiring a tissue graft. By thismethod the cellular suspension produced according to the first aspect ofthe invention is applied to a graft site. A liquid suspension containingcells may be manually distributed onto the graft site by any of severaltechniques, which include spraying, spreading, pipetting and painting.

[0064] In a highly preferred form of the invention the suspension issprayed on to a graft site. The suspension may be sprayed through anytype of nozzle that transforms liquid into small airborne droplets. Thisembodiment is subject to two constraints. First, it must not subject thecells in solution to shearing forces or pressures that would damage orkill substantial numbers of cells. Second, it should not require thatthe cellular suspension be mixed with a propellant fluid that is toxicor detrimental to cells or wound beds. A variety of nozzles that arecommonly available satisfy both constraints. Such nozzles may beconnected in any conventional way to a reservoir that contains thecellular suspension.

[0065] Alternatively the suspension may be delivered via a pipette,common “eye-droppers,” syringe and needle and or other similar devicesto place small quantities of cellular suspension on a graft site.

[0066] After the cell suspension has been applied to the recipient graftsite, the wound may be dressed with a wound dressing. In a preferredembodiment the dressing is Surfasoft™ a woven nylon dressing.Preferably, the healing of the wound is followed up by standardprotocols for skin graft treatment known to those skilled in the art.

[0067] According to the fourth aspect of the invention there is providedan apparatus for developing a tissue regeneration solution, which has aheating means suitable for heating an enzyme solution to a requiredtemperature and which is capable of maintaining that solution at thedesired temperature for a suitable amount of time; and a filter recesscomprising a filter means capable of separating large cellularcongregates from a cellular suspension.

[0068] In a preferred form of the fourth aspect of the invention theapparatus also includes a reservoir capable of holding a tissue sampleand a nutrient solution which solution is also capable of maintainingthe viability of the cells in the tissue sample. More preferably thereservoir is of sufficient size to permit manipulation of the tissuesample permitting separation of the tissue cellular stratum andharvesting of those cells from the stratum suitable for grafting.

[0069] The apparatus may also include one or more fluid containmentwells for storage of fluids such as the nutrient solution. The wells mayalternatively serve as a receptacle for a container such as a plastic orglass vial that holds the nutrient solution. Preferably, the well iscapable of holding at least a 10 ml volume. Such wells permit ease offluid application to the tissue sample. Storage of such fluids in closeproximity to its site of application also provides the advantage ofreducing the risk of accidental leakage of the fluid and provides aneasy means for accessing the fluid for accurately delivering it toeither the tissue sample or the cell suspension.

[0070] In a highly preferred form of the invention the apparatuscomprises a first and second member wherein:

[0071] (1) the first member includes:

[0072] (a) at least a heating means suitable for heating an enzymesolution to a required temperature and which is capable of maintainingthat solution at the desired temperature for a suitable amount of time;

[0073] (b) at least a filter recess comprising a filter means capable ofseparating large cellular congregates from a cellular suspension;

[0074] (c) at least a fluid containment well for storage of nutrientsolution;

[0075] (2) the second member forms a reservoir capable of withholding atissue sample and nutrient solution in fluid containment; and

[0076] wherein the first member provides a seat upon which the secondmember may be placed during manipulation of the tissue.

[0077] In a further preferred form of the invention the first memberprovides an storage compartment into which tools and solutions used inthe above described method may be stored. Where such a compartment isprovided in the apparatus the second member may provide the lid orclosure to that compartment. In use the lid is preferably removed fromthe top of the compartment and inverted. The underside of the lidpreferably forms the reservoir therein enabling the second member toserve a dual purpose. Tools and solutions used in the method can beaccessed from the compartment. The inverted lid may then be seated backover the compartment therein providing the reservoir for the apparatus.

[0078] The apparatus may be made from metal, plastic or any othermaterial. Further, the container may be any size. Preferably the size ofthe container is only limited by its intended use and the need forsterilisation such as by the use of gamma irradiation and or ethyleneoxide.

[0079] It should be appreciated that the heating means employed in theapparatus may simply constitute a heating pad or pads on the top of thefirst member. There are however, attendant problems with sucharrangements, not least of which is the possibility of accidentalspillage of the container undergoing heating. Therefore, in anembodiment, one or more heating means may be housed within a recess inthe first member. Also located within that recess is at least acontainer into which tissue may be placed for exposure to the enzymesolution. In an alternate embodiment one or more heating means may behoused in the base of the apparatus. In such a configuration the firstmember contains at least an opening suitable for receiving a containercapable of holding fluid, which opening provides access for thecontainer to the heating means.

[0080] It will be appreciated that if the apparatus is designed for morethan one use the heating means may be capable of being repeatedly heatedand cooled. Alternatively each heating unit may be capable of a singleuse, but multiple heating units may be provided with the apparatus tofacilitate multiple heating events.

[0081] In a highly preferred configuration of the apparatus a heatingcollar is located within a recess therein forming a heating recess inthe first member within which there is located a container (eg a vial)for the enzyme. The container is preferably held in place by at least arestraining means, which desirably surrounds part of the upper portionof the container preventing accidental release of the container from theapparatus. In circumstances, where the apparatus is intended for singleuse the restraining means may be formed as an integral part of the firstmember, thus meaning that removal of the container may only be achievedby physically breaking the first member.

[0082] The heating means used in the apparatus is preferably controlledby circuitry permitting activation of the heating element when required.For example the heating means may be switched on by depressing the startbutton located, for example, on the surface of the first member.Alternately the heating means may be activated by pushing the containerdown with sufficient force to activate a switch located in the base ofthe apparatus. A person of ordinary skill in the field will appreciatethat a wide range of electronic means may be used to activate theheating unit provided in the apparatus.

[0083] Desirably the heating unit is also operably linked to a timermechanism, which is adapted to heat the enzyme solution for apre-defined period of time. In circumstances where the apparatus isintended for multiple uses, preferably the timer can be set todeactivate the heating element when a particular amount of time isreached. At which point an alarm may activate to inform the user thatthe time is up. The alarm may be audible or in the form of a lightdisplay.

[0084] In a further preferred embodiment of the present invention theheating means may be provided with an adjustable temperature control.Where temperature adjustment is required such variation may be achievedby adapting the heating control circuitry to include or communicate witha temperature control mechanism permitting the temperature of theheating unit to be constantly varied within a constant range, or it maypresent a range of select temperatures that the heating control meanscan be set at. A temperature control means will beneficially be includedin the apparatus where the apparatus is to be used in the harvesting andpreparation of different cell types and or where different enzymes areused in the harvesting method for which the apparatus has uniqueapplication.

[0085] In an alternate more preferred form, the apparatus is designedfor single use. In such instances the timer mechanism is part of thecircuitry that controls the heating means. Once the heating means hasbeen activated it heats the solution for a predefined period of time andthen self-destructs. It should be appreciated by those skilled in theart that such an the apparatus may be fitted with various monitoringmeans that are capable of indicating such things as: the enzyme hasreached the required temperature; the amount of time that the enzyme hasbeen in the solution for; and or the amount of time left before thecircuitry self-destructs etc. By way of example only, the monitoringmeans might consist of a series of LED's that activate when certainevents occur. In a highly preferred embodiment the heating elementpreferably remains in the heating mode for a maximum of 45 minutes to 1hour.

[0086] The heating means may be powered by any means known in the art.Preferably, the power supply is provided by battery/batteries. In oneform of the invention, the power supply is a battery or a plurality ofbatteries located in the base of the apparatus.

[0087] In a further embodiment of the invention the apparatus may beprovided with one or more means to facilitate mixing of the solutionsused in the invention, such as for example an enzyme solution. In thisrespect, and by way of example only, the apparatus may include a meansfor vortexing the solution; such as an electromagnetic system that isadapted to agitate a magnetic bead. Where the apparatus includes anelectromagnetic mixing system the magnetic bead is preferably providedin the container (eg vial) in which the solution is stored in theapparatus. Alternatively the magnetic bead may be added to the solutionwhen mixing is desired.

[0088] In a highly preferred form of the invention the mixing means iscombined with the heating means either as a single unit or as separateunits to facilitate constant heating of the solution in an even manner.Using such a mixing means avoids the possible overheating of solutionclosest to the heating unit while the solution is heated. Such a systemwill provide a more constant heating of the solution. Alternate meansfor mixing the solution will be known in the art and include,mechanical, physical, electrical and electromagnetic means as anexample. While any mixing means may be employed in the apparatus,preferably the mixing means is either selected to minimise vibration ofthe apparatus or incorporated into the apparatus in a manner thatminimise such vibration. In this respect the mixing means may be housedon one or more vibration dampeners or the apparatus may include one ormore vibration dampeners on is base.

[0089] Where a mixing means is incorporated into the apparatus the meansmay be automatically activated upon activation of the heating unit oralternatively there may be a separate activation system. Further, thespeed of the mixing may either be fixed or variable. Preferably, thereis a separate activation system for the mixing means.

[0090] The filter recess incorporated into the apparatus may be of anysize or shape that facilitates filtering of a cellular suspension.Further the recess may be adapted to receive and hold at least a tubeinto which cell suspension may be filtered. Preferably the recess has aconical base providing easy means to access the full volume of cellsuspension after it has been filtered.

[0091] Desirably, the third recess is designed to receive a 100 μm cellfilter. The third recess can accommodate a 100 μm cell filter connectedto a conical tube. Preferably, the tube has area/volume graduationsmarked on the side.

[0092] The apparatus may also include a set of tools required for cellharvesting. It will be appreciated by those skilled in the art that anytools necessary for cell harvesting may be included with the device.Preferably, the set of tools are sterile. As an example only, the set oftools may include a glass vessel of separation enzyme; a sterilesolution for suspension of the enzyme; a sterile nutrient solution;scalpel; forceps; syringe; medicine dropper, cell filter; wounddressings and/or spray nozzles. In a highly preferred embodiment, theset of tools are stored in a compartment formed in the first member ofthe apparatus, which is covered by the second member when not in use.

[0093] In a highly preferred embodiment, the device is used to harvest asuspension of cells and apply the cells to a recipient site in thefollowing manner.

[0094] An aliquot of sterile water is mixed with a portion oflyophilised separation enzyme and placed in the heating recess. Theheating means is then activated which heats the contents (i.e. theenzyme solution) of the container to a working temperature of between 30and 37° C., preferably between 33 and 37° C. and by way of example 37°C. within 2 minutes and maintains the working temperature for at least45 minutes. Once an operational temperature has been reached, a sampleof tissue taken from a donor site is placed in the enzyme solution andincubated at the working temperature. The tissue sample is incubated forbetween 5 to 45 minutes. Those skilled in the art would appreciate thatthe time taken to achieve separation of the layers of the tissue sampleis dependent on the thickness and size of the tissue sample and theincubation temperature. Once enzymatic separation of the tissue layersis achieved, the tissue sample is removed to the reservoir and thetissue layers are separated using surgical instrument/s.

[0095] A carefully measured aliquot of the second solution is thenwithdrawn from the fluid containment well by aspiration into a syringeand then applied to the layers. The cells between the layers of tissueare scraped off and suspended by mixing with the nutrient solution. Thecell suspension is then collected, preferably using a syringe andcannula.

[0096] The harvested suspension of cells is then passed through a cellfilter located in the filter recess and the filtered suspension of cellsis collected into the filter recess. The reservoir may optionally berinsed with a further volume of the second solution and this resultingsuspension of cells also filtered and collected in the filter recess.

[0097] The filtered suspension of cells may then optionally be aspiratedinto a syringe and applied to the recipient site.

EXAMPLES

[0098] Further features of the present invention are more fullydescribed in the following non-limiting Examples. It is to beunderstood, however, that this detailed description is included solelyfor the purposes of exemplifying the present invention. It should not beunderstood in any way as a restriction on the broad description of theinvention as set out above.

[0099] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow the compositions and/or methods claimed herein are made andevaluated, and are intended to be purely exemplary of the invention andare not intended to limit the scope of what the inventors regard astheir invention. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. The present invention is moreparticularly described in the following examples which are intended asillustrative only since numerous modifications and variations thereinwill be apparent to those skilled in the art.

Example 1

[0100] Preparation of Recipient Site

[0101] To optimise the success of the skin graft, the wound was cleanedand assessed to be of the appropriate depth. Further, blood haemostasiswas established and the wound checked for evidence of surroundingcellulitis or infection. Techniques for preparing the area includedsharp dissection, dermabrasion or laser-resurfacing.

[0102] Donor Site Biopsy

[0103] The donor site was chosen to appropriately match the recipientsite. The donor site was infiltrated with local anaesthetic andadrenaline underneath the skin near the subcutaneous tissue. Thisallowed the donor site to be firm and aided in the taking a thinsplit-thickness biopsy. The dimensions of the biopsy were determined bythe size of the surface area of the recipient site to be covered.Typically, the biopsy size has an expansion ratio of 1:10-1:80. In thiscase, a biopsy size of 2 cm×2 cm was taken from the donor site giving anexpansion ratio of 1:60.

[0104] Cell Resurfacing Using the Rapid Technique

[0105] Treatment of the wound was carried out using the Re-Cell® RapidTechnique cell harvesting apparatus, which is explained in more detailin Example 2 below. The apparatus contained all the instruments,solutions, enzymes and dressings required for wound treatment.

[0106] The heating element was activated by depressing the “startbutton”. Solution (sterile water for injection) (10 ml) was transferredfrom the supplied plastic vessel marked Solution A into a glass vesselcontaining the separation enzyme (lyophilised trypsin) to give a finalconcentration of 0.5% trypsin. The enzyme solution was then mixedtogether, transferred to a vessel already located in the heating elementrecess and heated to 37° C.

[0107] The vessel containing Solution B (nutrient media) was transferredfrom its supplied vessel into the fluid containment well.

[0108] The previously obtained tissue sample was then placed in theenzyme solution and incubated at 37° C. for between 10 to 15 minutes.After this time, the tissue sample was removed from enzyme solution witha pair of forceps, rinsed by dipping into the fluid containment wellcontaining Solution B and placed with the dermal side down and theepidermal side up in the reservoir.

[0109] Solution B was then aspirated from the well into a syringe anddripped from the syringe onto both layers of the biopsy.

[0110] The skin layers were separated using forceps. This allowed accessto the zone of the dermal-epidermal junction of both surfaces. Cellswere scraped from the surfaces to develop a plume of cells in thereservoir. The cells were then mixed in Solution B. The plume of cellswas then drawn up into the syringe via a 19 gauge cannula.

[0111] The supplied filter (100 μm cell filter) was mounted in thefilter recess and the plume of cells in Solution B was passed throughthe filter. A further small amount of Solution B was then used to rinsethe reservoir (eg a petri dish) and collect any remaining cells, whichwere also passed through the filter.

[0112] The resulting suspension of cells collected in the conical recesswas aspirated into a syringe and a nozzle was attached to the syringefor spraying or dripping on to the wound area.

[0113] The wound was re-checked to ensure that it was clean and free ofdebris and that there was no evidence of bacterial contamination.Further, the wound was checked to determine if haemostasis had beenachieved. Once the recipient site was ready, the suspension of cells wasapplied to the wound surface using the nozzle.

[0114] The wound was dressed with Surfasoft™ a woven nylon dressing,which was supplied with the apparatus. The healing of the wound wasfollowed up using standard protocols for skin-graft treatment.

Example 2

[0115] The embodiment shown in FIG. 1 is directed to a Re-Cell® RapidTechnique cell harvesting apparatus 10 for use in producing atransplantable cellular suspension of living tissue suitable forgrafting to a patient.

[0116] As illustrated in FIG. 1 the apparatus includes a closure lid 12possessing a locking mechanism 14 adapted to releasably engage a baseportion 16. The locking mechanism 14 provides a means for closing theapparatus 16 when not in use. Located within the base portion 16 is afirst member 18 within which there is provided an aperture 20 in whichthere is located a vial 22 for the enzyme. Adjacent the aperture thereis provided an activation switch 24 capable of activating the heatingmeans (not shown). The first member also provides a fluid containmentwell 26 and a filter recess 28. As presented in this illustration thefilter 29 is shown located in the filter recess. Ordinarily the filteris an optional item included as a separate item in the apparatus.

[0117] The aperture 20 in the first member 18 is desirably of such adiameter that it allows the neck of the vial 22 to protrude through andabove the first member 18. The periphery of the aperture 20 is fittedwith a collar 21 which is slightly smaller than the diameter of the bodyof the vial 22. Thus, when in use, the vial 22 cannot be removed fromthe apparatus 10 as it is held in place by the collar 21 located aroundthe periphery of the aperture 20.

[0118] Located adjacent to the aperture 20, fluid containment well 26and filter recess 28 is the second member 30 which is positioned on aseat (not shown) located within a storage compartment (not shown) withinthe first member 18. When inverted the second member 30 forms areservoir within which tissue manipulations may be performed. Tofacilitate separation of the second member 30 from the first member 18an indent 32 is provided in the side of a portion of the second member30, which is of such a size that a person can lift the second memberfrom the seat on which it resides in the first member 18.

[0119] Within the filter recess 28 there is located a filter 29(provided separately with the other components) having a mesh thereincapable of separating cellular material of greater than 100 μm from acell supernatant.

[0120]FIG. 2 provides a partially exploded perspective view of theapparatus 10 wherein the second member 30 is removed from the firstmember 18 and inverted. Inversion of the second member 30 reveals thesidewalls 32 of the second member 30, which form the fluid containmentbarrier of the reservoir and a reservoir area 34 in which tissuemanipulations may be performed.

[0121] Removal of the second member 30 from the first member 18 alsoreveals a storage compartment 36 in the first member 18 in whichsolutions and tools may be stored when the apparatus 10 is not in use.Within the storage compartment 36 there is located a seat 38 upon whichthe second member 30 may reside. The seat 38 is preferably locatedaround the periphery of the storage compartment 36 at a depth beneaththe surface of the first member 18 that is equivalent to the height ofthe sidewalls 32 of the second member 30.

[0122]FIG. 3a provides a perspective view of the first member 18 showingthe storage compartment 36, the heater activating switch 24, theaperture 20, the fluid containment well 26 and filter recess 28 formedwithin the first member. FIG. 3b provides a rear view of the firstmember 18 showing the filter recess 28, the fluid containment well 26,the heater activating switch 24, the aperture collar 21 and the rearwall of the storage compartment 36. As seen in this figure the filterrecess has a conical base thereby providing a means for easy access tothe cell suspension that is filtered into it. Located adjacent to thefluid containment well and on the opposite side of the filtercontainment well there is also provided a battery positioning member 40which protrudes towards the base 16 of the apparatus (not shown) andprovides a means for holding the batteries in place, which are requiredfor activating the heating means.

[0123]FIG. 4 provides a perspective view of the base 16 of the apparatus10 showing the vial 22 located within a containment field 42. Betweenthe containment field 42 and the vial 22 there is located a heatingcollar(s) 44, which surrounds the body of the vial. Adjacent thecontainment field there is a circuit board 46, which is held in positionby circuit board containment means 48, 50 and 52. Said circuit board 46is in electrical communications with the heating collar(s) 44 by wires54. The circuit board is also in electrical communication via wires 58with the heater activating switch (not shown). Adjacent the circuitboard 46 there is provided a battery containment means 58, which holds 4AA batteries in immovable position (not shown). The batteries are inelectrical communication with the circuit board 46 by wires 60. When thefirst member 18 is fitted to the base 16 the batteries are held in placeby the battery containment means 58, the battery positioning means 40and the base of each of the fluid containment well 26 and the filterrecess 28. Preferably the conical base of the filter recess 28 alsoprotrudes between the batteries therein providing a further means forsecuring the batteries in immovable position.

[0124] Modifications and variations of the described methods and deviceof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in the relevant field inwhich this invention resides are intended to be within the scope of thefollowing claims.

What is claimed is:
 1. A method for preparing a cell suspension suitablefor application to a patient, which method comprises the steps of: (a)subjecting a tissue sample including cells suitable for grafting to apatient, to at least a physical and or chemical dissociating meanscapable of dissociating cellular stratum in the tissue sample; (b)removing the tissue sample from the dissociating means used in step (a)and harvesting in the presence of a nutrient solution cells from thetissue sample, cells suitable for grafting on to a patient wherein thenutrient solution is (i) free of xenogenic serum, (ii) capable ofmaintaining the viability of the cells until applied to a patient and(iii) is suitable for direct application to a region on a patientundergoing tissue grafting; and (c) filtering the cellular suspensionproduced according to step (b) to remove large cellular conglomerates.2. A method according to claim 1 wherein the enzyme suitable fordissociating cohesive pieces of tissue stratum in the sample is trypsinor a trypsin-like enzyme.
 3. A method according to claim 2 wherein theenzyme is selected from the group consisting of trypsin, trypsin-EDTA,dispase, collagenase, thermolysin, pronase, hyaluronidase, pancreatin,elastase and papain.
 4. A method according to claim 1 wherein thenutrient solution is Hartmann's solution.
 5. A cell suspension producedaccording to the method of claim
 1. 6. A cell suspension according toclaim 5 prepared from autologous cells.
 7. A method of treating apatient in need of graft surgery, said method comprising the steps of:(a) preparing a cell suspension according to the method of claim1; and(b) administering the suspension directly to a region on the patientthat requires a cell graft in a manner that facilitates spreading of thecell suspension in a relatively even distribution over the graft region.8. Use of a cellular suspension suitable for grafts, which suspension isprepared according to the following steps: (a) subjecting a tissuesample including cells suitable for grafting to a patient, to an enzymesuitable for dissociating cohesive pieces of the tissue stratum in thesample; (b) removing the sample from the enzyme solution used in step(a) and harvesting in the presence of a nutrient solution cells from thetissue sample, which cells are suitable for grafting on to a patientwherein the nutrient solution is (i) free of xenogenic serum, (ii)capable of maintaining the viability of the cells until applied to apatient and (iii) is suitable for direct application to a region on apatient undergoing tissue grafting; and (c) filtering the cellularsuspension produced according to step (b) to remove large cellularconglomerates; for the preparation of therapeutic preparation suitablefor the treatment of tissue disorders requiring grafting.
 9. The useaccording to claim 8 wherein the nutrient solution is Hartmann'ssolution.
 10. An apparatus for developing a tissue regenerationsolution, said apparatus comprising: (a) a heating means suitable forheating an enzyme solution to a required temperature and which iscapable of maintaining that solution at the desired temperature for asuitable amount of time; and (b) a filter recess comprising a filtermeans capable of separating large cellular congregates from a cellularsuspension.
 11. The apparatus according to claim 10, which additionallycomprises a reservoir capable of holding a tissue sample and a nutrientsolution.
 12. The apparatus according to claim 10, which includes one ormore fluid containment wells for storage of fluids.
 13. An apparatus fordeveloping a tissue regeneration solution, comprising a first and secondmember wherein: (i) the first member includes: (a) a heating meanssuitable for heating an enzyme solution to a required temperature andwhich is capable of maintaining that solution at the desired temperaturefor a suitable amount of time; (b) a filter recess comprising a filtermeans capable of separating large cellular congregates from a cellularsuspension; (c) at least a fluid containment well for storage ofnutrient solution; (ii) the second member forms a reservoir capable ofwithholding a tissue sample and nutrient solution in fluid containment;and wherein the first member provides a seat upon which the secondmember may be placed during manipulation of the tissue.